Bottom Line:
Consequently, both in locomoting and stationary cells, myosin clusters approached the cell body boundary, where they became compressed and aligned, resulting in the formation of boundary bundles.In locomoting cells, the compression was associated with forward displacement of myosin features.These data are not consistent with either sarcomeric or polarized transport mechanisms of cell body translocation.

ABSTRACTWhile the protrusive event of cell locomotion is thought to be driven by actin polymerization, the mechanism of forward translocation of the cell body is unclear. To elucidate the mechanism of cell body translocation, we analyzed the supramolecular organization of the actin-myosin II system and the dynamics of myosin II in fish epidermal keratocytes. In lamellipodia, long actin filaments formed dense networks with numerous free ends in a brushlike manner near the leading edge. Shorter actin filaments often formed T junctions with longer filaments in the brushlike area, suggesting that new filaments could be nucleated at sides of preexisting filaments or linked to them immediately after nucleation. The polarity of actin filaments was almost uniform, with barbed ends forward throughout most of the lamellipodia but mixed in arc-shaped filament bundles at the lamellipodial/cell body boundary. Myosin II formed discrete clusters of bipolar minifilaments in lamellipodia that increased in size and density towards the cell body boundary and colocalized with actin in boundary bundles. Time-lapse observation demonstrated that myosin clusters appeared in the lamellipodia and remained stationary with respect to the substratum in locomoting cells, but they exhibited retrograde flow in cells tethered in epithelioid colonies. Consequently, both in locomoting and stationary cells, myosin clusters approached the cell body boundary, where they became compressed and aligned, resulting in the formation of boundary bundles. In locomoting cells, the compression was associated with forward displacement of myosin features. These data are not consistent with either sarcomeric or polarized transport mechanisms of cell body translocation. We propose that the forward translocation of the cell body and retrograde flow in the lamellipodia are both driven by contraction of an actin-myosin network in the lamellipodial/cell body transition zone.

Figure 11: Models of cell body translocation. Actin filaments are shown as gray lines, and myosin filaments are shown as black lines or dumbbell figures. (I) The sarcomeric mechanism cannot drive the cell body forward because arc-shaped actin– myosin II bundles, upon contraction, would produce a backward-directed net force on the cell body. (II) The transport model does not fit the data because myosin clusters in the lamellipodia, the only part of a cell where transport tracks are present, do not move forward. (III) Forward rolling driven by actin–myosin axles seems problematic because of the predominantly backward orientation of flanking bundles (a, top view). These bundles may participate in rolling by exerting a rearward-directed force at the bottom (b, vertical section); however, the origin of forward-directed force remains unclear (question mark). (IV) According to the dynamic network model, contraction of an actin–myosin network in the lamellipodia– cell body transition zone is coupled to forward translocation. (a) In the lamellipodium, the network of divergent actin filaments interacts with clusters of myosin bipolar filaments. Whereas small myosin clusters situated in the dense network close to cell front cannot move, bigger clusters in the sparser network in the transition zone are capable of approaching the barbed ends of diverging filaments and moving forward. (b) As myosin clusters move forward, they align actin filaments parallel to the leading edge. (c) Overall, network contraction at the lamellipodia–cell body transition zone results in formation of actin–myosin bundles and forward translocation of the cell body. (d) Vertical section view shows that forward rolling would result from a combination of network contraction in front of the cell body with rearward drag resulting from actin–myosin bundles at the bottom of the body.

Mentions:
This model implies the existence of actin–myosin II bundles organized in a semisarcomeric fashion and contracting during locomotion. In keratocytes, the only prominent actin filament bundles possibly organized this way were arc-shaped bundles at the cell body boundary. However, they were oriented perpendicular to the direction of cell locomotion and, therefore, not likely to provide the driving force. Moreover, since these bundles were arc shaped, with their convex side forward and presumably attached strongest at their trailing lateral edges (as suggested by wrinkling of elastic substrata [Lee et al., 1994]), their contraction would create a force component in the direction opposite to the direction of locomotion (Fig. 11 I).

Figure 11: Models of cell body translocation. Actin filaments are shown as gray lines, and myosin filaments are shown as black lines or dumbbell figures. (I) The sarcomeric mechanism cannot drive the cell body forward because arc-shaped actin– myosin II bundles, upon contraction, would produce a backward-directed net force on the cell body. (II) The transport model does not fit the data because myosin clusters in the lamellipodia, the only part of a cell where transport tracks are present, do not move forward. (III) Forward rolling driven by actin–myosin axles seems problematic because of the predominantly backward orientation of flanking bundles (a, top view). These bundles may participate in rolling by exerting a rearward-directed force at the bottom (b, vertical section); however, the origin of forward-directed force remains unclear (question mark). (IV) According to the dynamic network model, contraction of an actin–myosin network in the lamellipodia– cell body transition zone is coupled to forward translocation. (a) In the lamellipodium, the network of divergent actin filaments interacts with clusters of myosin bipolar filaments. Whereas small myosin clusters situated in the dense network close to cell front cannot move, bigger clusters in the sparser network in the transition zone are capable of approaching the barbed ends of diverging filaments and moving forward. (b) As myosin clusters move forward, they align actin filaments parallel to the leading edge. (c) Overall, network contraction at the lamellipodia–cell body transition zone results in formation of actin–myosin bundles and forward translocation of the cell body. (d) Vertical section view shows that forward rolling would result from a combination of network contraction in front of the cell body with rearward drag resulting from actin–myosin bundles at the bottom of the body.

Mentions:
This model implies the existence of actin–myosin II bundles organized in a semisarcomeric fashion and contracting during locomotion. In keratocytes, the only prominent actin filament bundles possibly organized this way were arc-shaped bundles at the cell body boundary. However, they were oriented perpendicular to the direction of cell locomotion and, therefore, not likely to provide the driving force. Moreover, since these bundles were arc shaped, with their convex side forward and presumably attached strongest at their trailing lateral edges (as suggested by wrinkling of elastic substrata [Lee et al., 1994]), their contraction would create a force component in the direction opposite to the direction of locomotion (Fig. 11 I).

Bottom Line:
Consequently, both in locomoting and stationary cells, myosin clusters approached the cell body boundary, where they became compressed and aligned, resulting in the formation of boundary bundles.In locomoting cells, the compression was associated with forward displacement of myosin features.These data are not consistent with either sarcomeric or polarized transport mechanisms of cell body translocation.

ABSTRACTWhile the protrusive event of cell locomotion is thought to be driven by actin polymerization, the mechanism of forward translocation of the cell body is unclear. To elucidate the mechanism of cell body translocation, we analyzed the supramolecular organization of the actin-myosin II system and the dynamics of myosin II in fish epidermal keratocytes. In lamellipodia, long actin filaments formed dense networks with numerous free ends in a brushlike manner near the leading edge. Shorter actin filaments often formed T junctions with longer filaments in the brushlike area, suggesting that new filaments could be nucleated at sides of preexisting filaments or linked to them immediately after nucleation. The polarity of actin filaments was almost uniform, with barbed ends forward throughout most of the lamellipodia but mixed in arc-shaped filament bundles at the lamellipodial/cell body boundary. Myosin II formed discrete clusters of bipolar minifilaments in lamellipodia that increased in size and density towards the cell body boundary and colocalized with actin in boundary bundles. Time-lapse observation demonstrated that myosin clusters appeared in the lamellipodia and remained stationary with respect to the substratum in locomoting cells, but they exhibited retrograde flow in cells tethered in epithelioid colonies. Consequently, both in locomoting and stationary cells, myosin clusters approached the cell body boundary, where they became compressed and aligned, resulting in the formation of boundary bundles. In locomoting cells, the compression was associated with forward displacement of myosin features. These data are not consistent with either sarcomeric or polarized transport mechanisms of cell body translocation. We propose that the forward translocation of the cell body and retrograde flow in the lamellipodia are both driven by contraction of an actin-myosin network in the lamellipodial/cell body transition zone.